Synapsin II Regulation of GABAergic Synaptic Transmission Is Dependent on Interneuron Subtype

Abstract

Synapsins are epilepsy susceptibility genes that encode phosphoproteins reversibly associated with synaptic vesicles. Synapsin II (SynII) gene deletion produces a deficit in inhibitory synaptic transmission, and this defect is thought to cause epileptic activity. We systematically investigated how SynII affects synchronous and asynchronous release components of inhibitory transmission in the CA1 region of the mouse hippocampus. We found that the asynchronous GABAergic release component is diminished in SynII-deleted (SynII(-)) slices. To investigate this defect at different interneuron subtypes, we selectively blocked either N-type or P/Q-type Ca²⁺ channels. SynII deletion suppressed the asynchronous release component at synapses dependent on N-type Ca²⁺ channels but not at synapses dependent on P/Q-type Ca²⁺ channels. We then performed paired double-patch recordings from inhibitory basket interneurons connected to pyramidal neurons and used cluster analysis to classify interneurons according to their spiking and synaptic parameters. We identified two cell subtypes, presumably parvalbumin (PV) and cholecystokinin (CCK) expressing basket interneurons. To validate our interneuron classification, we took advantage of transgenic animals with fluorescently labeled PV interneurons and confirmed that their spiking and synaptic parameters matched the parameters of presumed PV cells identified by the cluster analysis. The analysis of the release time course at the two interneuron subtypes demonstrated that the asynchronous release component was selectively reduced at SynII(-) CCK interneurons. In contrast, the transmission was desynchronized at SynII(-) PV interneurons. Together, our results demonstrate that SynII regulates the time course of GABAergic release, and that this SynII function is dependent on the interneuron subtype.SIGNIFICANCE STATEMENT Deletion of the neuronal protein synapsin II (SynII) leads to the development of epilepsy, probably due to impairments in inhibitory synaptic transmission. We systematically investigated SynII function at different subtypes of inhibitory neurons in the hippocampus. We discovered that SynII affects the time course of GABA release, and that this effect is interneuron subtype specific. Within one of the subtypes, SynII deficiency synchronizes the release and suppresses the asynchronous release component, while at the other subtype SynII deficiency suppresses the synchronous release component. These results reveal a new SynII function in the regulation of the time course of GABA release and demonstrate that this function is dependent on the interneuron subtype.

Keywords: CCK; GABA; PV; epilepsy; hippocampus; inhibitory.

Figure 1.

The asynchronous release component is reduced at SynII(−) inhibitory hippocampal synapses. A, Basal inhibitory transmission is similar at WT and SynII(−) slices. The stimulation intensity was adjusted to eliminate transmission failures, and this stimulation paradigm produced similar IPSC amplitudes in WT in SynII(−) lines. B, Representative postsynaptic currents elicited by 50 Hz frequency tetani at WT and SynII(−) slices illustrate the reduced after tetanus current in SynII(−) line (red). Stimulus artifacts were removed for clarity. C, The relationship between the charge transfer after a tetanus and the charge during a tetanus at individual experiments. The slope of the linear regression is significantly reduced at SynII(−) slices (p < 0.01), suggesting a selective reduction of the charge transfer after the tetanus in the Syn II(−) line. D, The After/During charge ratio is significantly reduced at SynII(−) slices (p < 0.001). E, The frequency of asynchronous current peaks is significantly reduced at SynII(−) slices (p < 0.005), whereas the sIPSC frequency is not affected. sIPSCs were collected for 5 min before the onset of the stimulation. F, The synchronous release component at WT and SynII(−) slices over the duration of the tetanus computed using deconvolution. G, The cumulative synchronous release kinetics derived by deconvolution suggest that the synchronous release component is not affected at SynII(−) slices. Dotted lines indicate confidence interval for the cumulative synchronous release. Inset, Total synchronous release over the duration of the tetanus. H, The asynchronous release component computed using deconvolution. Right, Plot (overlay) represents the data normalized by the first response in a tetanus. I, The cumulative asynchronous release component is significantly reduced at SynII(−) slices. Dotted lines indicate the confidence interval. Inset, Total asynchronous release, including that recorded during and after the tetanus (p < 0.01). J, The ratio between the cumulative asynchronous and synchronous release components is significantly reduced at SynII(−) slices (p < 0.01). Data collected from 26 slices for each genotype. **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 2.

The blockade of N-type Ca²⁺ channels by ω-conotoxin GVIA reverses the inhibitory effect of SynII deletion on the asynchronous GABAergic release component. A, The blockade of N-type Ca²⁺ channels by ω-conotoxin GVIA (250 nm) significantly reduces the basal inhibitory transmission in both genotypes. B, Representative current traces elicited by 50 Hz tetani illustrate the reduced postsynaptic signal in both lines upon ω-conotoxin GVIA application. Notably, the effect of the treatment on the after tetanus current is stronger in WT slices. Stimulus artifacts are removed for clarity. C, The ω-conotoxin GVIA treatment significantly (p < 0.005) reduces the frequency of asynchronous peaks at WT but not at SynII(−) slices. D, The effect of ω-conotoxin GVIA on the charge transfer during the tetanus is similar at WT and SynII(−) slices. E, The effect of ω-conotoxin GVIA on the charge transfer after the tetanus is significantly stronger at WT slices (p < 0.01). F, The ω-conotoxin GVIA treatment reduces the After/During charge ratio at WT (p < 0.005) but not at SynII(−) slices. Data collected from 13 slices for each genotype. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 3.

The blockade of P/Q-type Ca²⁺ channels by ω-agatoxin suppresses both release components in WT and SynII(−) genotypes. A, The blockade of P/Q-type Ca²⁺ channels by ω-agatoxin IVA (10 nm) significantly reduces basal inhibitory transmission in both genotypes. B, Representative traces illustrate a uniform reduction of the postsynaptic current produced by the ω-agatoxin IVA treatment in both genotypes. C, The ω-agatoxin IVA treatment does not affect the frequency of asynchronous peaks in either of the genotypes. D, The effect of ω-agatoxin IVA on the charge transfer during the tetanus is similar at WT and SynII(−) slices. E, The effect of ω-agatoxin IVA on the charge transfer after the tetanus is similar at WT and SynII(−) slices. F, The ω-agatoxin IVA treatment does not affect the After/During charge ratio in either of the lines. Data collected from 13 slices for each genotype. *p < 0.05, **p < 0.01.

Figure 4.

Paired recordings from inhibitory basket cells connected to CA1 neurons at the stratum pyramidale reveal two classes of interneurons with fast and slow IPSC kinetics. A, Paired recordings from GABAergic basket cells connected to pyramidal neurons. B, A GABAergic basket cell (red) synaptically connected to a pyramidal neuron (blue). Biotin staining shows axon arbors predominantly located in stratum pyramidale (SP). SO, Stratum oriens; s.rad, stratum radiatum; SLM, stratum lacunosum-moleculare. C, All the examined connected pairs fall into two clusters according to the rise and decay times of the recorded IPSCs. Examples of IPSCs with fast and slow kinetics recorded from different interneuron types are shown on the left.

Figure 5.

Perisomatic interneurons with fast IPSC kinetics constitute two interneuron subtypes, FS and RS. All the connected pairs are classified according to five parameters: SF (A), CA (B), CV (C), FR (C), and PPR (D). *Significant difference (p < 0.05) between the interneuron subtypes (two-way ANOVA). A, Spiking frequencies differ between neuron subtypes but not between the genotypes. B, RS interneurons have a lower CA, which is manifested as a stronger decay in SF. C, RS interneurons have a higher variability in IPSC amplitudes, CV, and a higher failure rate. D, RS interneurons have a higher PPR. E, A 3D plot showing that all the interneuron subtypes fell into two distinct clusters according to three physiological parameters: SF, CA, and PPR. WT and SynII(−) genotypes were pooled together because all the plotted parameters were similar for WT and SynII(−) lines.

Figure 6.

Spiking and synaptic parameters of FS basket interneurons match the parameters obtained for PV⁺ basket interneurons. A, The layer of labeled PV cells in the CA region of the hippocampus. B, Paired recordings from PV⁺ interneurons connected to pyramidal neurons. Top to bottom, DIC image showing the recording configuration, an epifluorescence image of the same field of view showing the presynaptic PV⁺ cell, and an overlay. C, IPSC amplitudes are similar for PV⁺ and FS cells. D, Spiking frequencies are similar for PV⁺ and FS cells. E, CAs are similar for PV⁺ and FS cells. F, Coefficients of variation are similar for PV⁺ and FS cells. G, PPRs are similar for PV⁺ and FS cells. H, The 3D plot depicting the three parameters, CF, CA, and PPR, shows that PV⁺ and FS interneurons fall within the same cluster. Data collected from nine PV⁺ cells.

Figure 7.

SynII deletion suppresses the asynchronous release component at regular-spiking (presumed CCK) interneurons. A, Representative traces illustrating that the after tetanus current is decreased at SynII(−) neurons. B, Asynchronous IPSC frequency is significantly (p < 0.05) reduced at SynII(−) neurons, whereas the sIPSC frequency is not affected. Left, Representative traces show asynchronous and spontaneous IPSCs. C, Cumulative probability plots show that SynII has no effect on the charge transfer during the tetanus (left), whereas the charge transfer after the tetanus is significantly reduced at SynII(−) neurons (middle, the distribution is shifted to the left, p < 0.05, Kolomogorov–Smirnov test). The After/During charge ratio is significantly decreased at SynII(−) neurons (right, p < 0.05). D, The synchronous release component at WT and SynII(−) neurons computed using the deconvolution. Right, Plot represents data normalized by the first response in a tetanus. E, The asynchronous release component computed using deconvolution. Right, Plot represents data normalized by the first response in a tetanus. F, The cumulative asynchronous release component is significantly decreased at SynII neurons (p < 0.05). Dotted lines indicate the confidence interval. G, The cumulative synchronous release component is significantly increased at SynII neurons (p < 0.05). Dotted lines indicate the confidence interval. H, The ratio between the cumulative asynchronous and synchronous release components is reduced at SynII(−) neurons (p < 0.05). Data collected from six pairs for each genotype. *p < 0.05.

Figure 8.

SynII deletion suppresses the synchronous release component and enhances the asynchronous release component at FS (PV) interneurons. A, Representative traces illustrate a mild selective increase in the after tetanus current in the SynII(−) line. B, The frequency of asynchronous IPSCs is significantly (p < 0.05) increased at SynII(−) neurons, whereas the sIPSC frequency is not affected. Left traces, Examples of asynchronous and spontaneous IPSCs. C, The charge transfer after the tetanus is selectively increased at SynII(−) neurons. This is evident from the right shift in the cumulative distribution representing the after tetanus charge (p < 0.05, Kolomogorov–Smirnov test) and a significant increase in the After/During charge ratio at SynII(−) neurons (p < 0.05). D, The synchronous release component at WT and SynII(−) KO interneurons computed using deconvolution. E, The asynchronous release component computed using deconvolution. Right plot, Data normalized by the first response in a tetanus. F, The cumulative asynchronous release component is significantly increased at SynII neurons (p < 0.05). Dotted lines indicate the confidence interval. G, The cumulative synchronous release component is significantly decreased at SynII neurons (p < 0.05). Dotted lines indicate the confidence interval. H, The ratio between the total asynchronous and synchronous release components is significantly increased at SynII(−) neurons (p < 0.05). Data collected from six pairs for each genotype. *p < 0.05.

Figure 9.

SynII deletion desynchronizes basal transmission at regular spiking (CCK) interneurons and synchronizes transmission at FS (PV) interneurons. A, Representative traces illustrate higher IPSC amplitudes and faster kinetics at RS SynII(−) neurons. B, The average IPSC traces normalized by the peak amplitudes demonstrate a narrower time course at the currents recorded from FS SynII(−) pairs. C, The normalized cumulative charge plot shows a significantly faster IPSC kinetics at the SynII(−) line (p < 0.05, Kolomogorov–Smirnov test). D–F, At FS interneurons, SynII deletion produces a slower IPSC kinetics. G, SynII deletion increases the IPSC amplitude at RS interneurons and decreases the IPSC amplitude at FS interneurons (p < 0.05, one-way ANOVA followed by the Tukey's test). H, SynII(−) deletion does not affect the IPSC charge at either of the interneuron subtypes, suggesting that the release magnitude is not affected. I, The ratio between the IPSC charge and amplitude, as a measure of IPSC kinetics. SynII(−) deletion significantly reduces the ratio at RS interneurons and significantly increases the ratio at FS interneurons (p < 0.05, one-way ANOVA followed by the Tukey's test), suggesting that SynII regulates the release kinetics at FS and RS interneurons in opposite ways. *p < 0.05.